title: Modified house-keeping gene expression in a rat tail compression loading-induced disc degeneration model.
abstract: House-keeping genes (HKGs) are generally used as endogenous controls for molecular normalization in quantitative PCR analysis. However, whether all the so-called HKGs are useful for intervertebral disc research is controversial. Our objective was, using a prevalidated rat tail static compression loading-induced disc degeneration model, to clarify the feasibility of common HKGs for gene-quantification in the nucleus pulposus cells. In real-time RT-PCR for five HKGs [β-actin, β-glucuronidase, β-2 microglobulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and lactate dehydrogenase A (LDHA)], static compression at 1.3 MPa for up to 56 days demonstrated messenger RNA (mRNA) expression levels of consistent β-2 microglobulin and GAPDH, slightly up-regulated β-glucuronidase, and fairly down-regulated β-actin and LDHA. Especially, β-actin had a drastic suppression to 0.15-fold in the loaded relative to unloaded discs at 7 days. In immunofluorescence, β-actin showed a significant down-regulation to almost undetectable levels from 28 days, while GAPDH was constantly detected throughout. β-Actin mRNA and protein-distribution are thought to be affected by the loading treatment, however, GAPDH mRNA and protein-distribution can retain relatively stable expressions. Under prolonged static compression, β-actin and probably LDHA are inappropriate, and GAPDH is a feasible HKG as internal references in the disc cells.
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yurube
authors.forename: Toru
authors.initials: T
authors.lastname: Takada
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Hirata
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Kakutani
authors.forename: Koichiro
authors.initials: K
authors.lastname: Maeno
authors.forename: Zhongying
authors.initials: Z
authors.lastname: Zhang
authors.forename: Junya
authors.initials: J
authors.lastname: Yamamoto
authors.forename: Minoru
authors.initials: M
authors.lastname: Doita
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kurosaka
authors.forename: Kotaro
authors.initials: K
authors.lastname: Nishida
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D005455:Fluorescent Antibody Technique; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000235:genetics / Q000378:metabolism; D007211:Indoles; D055959:Intervertebral Disc Degeneration / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D000080864:Lactate Dehydrogenase 5; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013314:Stress, Mechanical; D001613:beta 2-Microglobulin / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; D007211:Indoles; D007527:Isoenzymes; D001613:beta 2-Microglobulin; C007293:DAPI; D007770:L-Lactate Dehydrogenase; D000080864:Lactate Dehydrogenase 5; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); D005966:Glucuronidase